Autoproteolysis of PIDD marks the bifurcationbetween pro-death caspase-2 and pro-survivalNF-jB pathway

Antoine Tinel1,3, Sophie Janssens1,4,Saskia Lippens1, Solange Cuenin1,Emmanuelle Logette1, BastienneJaccard1,2, Manfredo Quadroni1,2

and Jurg Tschopp1,*1Department of Biochemistry, University of Lausanne, Epalinges,Switzerland and 2Protein Analysis Facility, University of Lausanne,Epalinges, Switzerland

Upon DNA damage, a complex called the PIDDosome is

formed and either signals NF-jB activation and thus cell

survival or alternatively triggers caspase-2 activation and

apoptosis. PIDD (p53-induced protein with a death do-

main) is constitutively processed giving rise to a 48-kDa

N-terminal fragment containing the leucine-rich repeats

(LRRs, PIDD-N) and a 51-kDa C-terminal fragment con-

taining the death domain (DD, PIDD-C). The latter under-

goes further cleavage resulting in a 37-kDa fragment

(PIDD-CC). Here we show that processing occurs at S446

(generating PIDD-C) and S588 (generating PIDD-CC) by

an auto-processing mechanism similar to that found in the

nuclear pore protein Nup98/96 and inteins. Auto-cleavage

of PIDD determines the outcome of the downstream

signaling events. Whereas initially formed PIDD-C medi-

ates the activation of NF-jB via the recruitment of RIP1

and NEMO, subsequent formation of PIDD-CC causes cas-

pase-2 activation and thus cell death. A non-cleavable

PIDD mutant is unable to translocate from the cytoplasm

to the nucleus and loses both activities. In this way, auto-

proteolysis of PIDD might participate in the orchestration

of the DNA damage-induced life and death signaling

pathways.

The EMBO Journal (2007) 26, 197–208. doi:10.1038/

sj.emboj.7601473; Published online 7 December 2006

Subject Categories: proteins; differentiation & death

Keywords: auto-proteolysis; caspase-2; DNA damage; NF-kB;

PIDD

Introduction

PIDD (p53-induced protein with a death domain), also

known as LRDD (leucine-rich repeat and death domain-

containing protein), was discovered during a bioinformatics

screen for novel death domain (DD)-containing proteins

using the DD of RIP1 as bait (Telliez et al, 2000).

Independently, PIDD was identified as a novel p53-inducible

gene in an erythroleukemia cell line expressing a tempera-

ture-sensitive p53 mutant allele, and shown to be implicated

in p53-dependent apoptosis (Lin et al, 2000). PIDD contains

seven leucine-rich repeats (LRRs) at the N-terminus, followed

by two ZU-5 domains and a C-terminal DD.

Recent studies have shown that PIDD plays a critical role in

the activation of caspase-2 (Tinel and Tschopp, 2004; Berube

et al, 2005; Ren et al, 2005; Seth et al, 2005). As caspase-2

shares characteristics of both effector and initiator caspases,

its activation mechanism remained enigmatic for a long time.

An important breakthrough was made by showing that

caspase-2 was activated in a high-molecular-weight complex,

reminiscent of the apoptosome and other initiator caspase-

activating platforms (Read et al, 2002). The recruitment and

activation of caspase-2 within this complex is entirely depen-

dent on the presence of an adaptor protein called RAIDD and

on PIDD, which together with caspase-2 constitute the so-

called PIDDosome (Tinel and Tschopp, 2004; Berube et al,

2005). These results were particularly intriguing as PIDD, just

like caspase-2, had been suspected to participate in DNA

damage-induced apoptosis (Robertson et al, 2002; Castedo

et al, 2004; Giagkousiklidis et al, 2005; Zhivotovsky and

Orrenius, 2005; Vakifahmetoglu et al, 2006).

PIDD also plays an essential role in NF-kB activation in-

duced by DNA damage. Indeed, genotoxic stress triggers the

formation of an alternative PIDDosome, consisting of PIDD,

RIP1 and NEMO (Hur et al, 2003; Janssens et al, 2005). NEMO

is recruited to the PIDDosome via RIP1 and appears to be

modified immediately upon recruitment to the activated

PIDDosome by sumoylation (Janssens et al, 2005). This is

the beginning of a sequential series of modifications on

NEMO, in which the nuclear protein kinase ATM plays an

essential role, and which alter the protein’s subcellular locali-

zation, providing a means to link a nuclear signal (DNA

damage) to the activation of a cytosolic NF-kB-activating

complex (the IKK complex) (Huang et al, 2003; Wu et al, 2006).

Regulation of protein activation can be achieved in many

different ways. Apart from a tightly controlled transcriptional

response as a DNA damage-dependent response gene, PIDD

activation appears to be controlled by a second, unusual

mechanism of post-translational cleavage. Full-length PIDD

has a predicted molecular mass of 100 kDa, but is constitu-

tively processed at two sites giving rise to an N-terminal

fragment of 48 kDa and a C-terminal fragment of 51 kDa,

which is further cleaved, resulting in a 37 kDa fragment

(Telliez et al, 2000; Tinel and Tschopp, 2004). The current

study aims at characterizing the molecular mechanisms

underlying PIDD cleavage and determining its functional

consequences.Received: 29 June 2006; accepted: 6 November 2006; publishedonline: 7 December 2006

*Corresponding author. Department of Biochemistry, University ofLausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland.Tel.: þ 41 21 692 5738; Fax: þ 41 21 692 6705;E-mail: jurg.tschopp@unil.ch3Present address: Department of Pathology, Harvard Medical School,77 avenue Louis Pasteur, Boston, MA 02115, USA4Present address: Department of Molecular Biomedical Research,University of Ghent-VIB, Technologiepark 927, 9052 Gent, Belgium

The EMBO Journal (2007) 26, 197–208 | & 2007 European Molecular Biology Organization | All Rights Reserved 0261-4189/07

www.embojournal.org

&2007 European Molecular Biology Organization The EMBO Journal VOL 26 | NO 1 | 2007

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Results

PIDD is constitutively processed at S446 and S588

As previously reported, full-length PIDD (PIDD-FL, 910 aa)

with an apparent molecular weight of 100 kDa is constitu-

tively cleaved into three main fragments, an N-terminal

fragment of approximately 48 kDa (PIDD-N) and two

C-terminal fragments of 51 kDa (PIDD-C) and 37 kDa (PIDD-

CC) (Tinel and Tschopp, 2004). This processing is well

documented for cells in which PIDD is transiently or stably

overexpressed. In order to exclude an artifact of overexpres-

sion, it was however important to determine whether these

cleavage events also occurred at the level of the endogenous

protein. A monoclonal antibody directed against the DD of

PIDD was generated, which was able to detect endogenous

PIDD. In all cell lines tested, the PIDD-C and the PIDD-CC

fragments formed the major species, whereas full-length

endogenous PIDD was hardly detectable or even absent

(Figure 1A and Supplementary Figure 1A). Interestingly,

several species were detected around 51 kDa, possibly reflect-

ing the expression of different isoforms, as all species dis-

appeared when the cells were transfected with siRNA directed

against PIDD (Supplementary Figure 1B). The ratio between

the PIDD-C and PIDD-CC fragments varied from cell line to

A

B

Moc

k

Fla

g-P

IDD

PIDD-FL

PIDD-C

PIDD-CC

S446

910778 873124 285 320 420 455 554

DDZU-5ZU-5

LRR

1 PIDD-FL

910DDZU-5

S588

PIDD-C446

910DD PIDD-CC588

ZU-51 445 PIDD-N

WB: αPIDD

ID

D

C

PIDD-C

PIDD-FL

PIDD-CC

Moc

k

PID

D

PID

DS

446A

PID

DS

588A

PID

DS

446A

/S58

8A

WB: αFlag

WB: αRAIDD

PIDD-FL

Raj

i B c

ells

Ram

os B

cel

ls

Jurk

at T

cel

ls

293T

HC

T11

6

U2O

S

83 -62 -

47 -

HCT116 p53

+/+ –/–

WB: αPIDD

WB: αRAIDD

WB: αCasp2

PIDD-C

PIDD-CC

83 -

62 -

47 -

32 -

Moc

k

PID

D

IP: αVSV

WB: αVSV

Moc

k

PID

D

Lysates

83 -

62 -

47 -

32 -

Moc

k

PID

D

IP: αFlag

IP: αVSV

IP: αVSV

WB: αFlag

WB: αFlag

WB: αVSV

WB: αFlag

Moc

k

PID

D

Lysates

PIDD-C

PIDD-FL

PIDD-CC

PIDD-N

E

PIDD-C

PIDD-CC

PIDD-CCPIDD-CS588A

PIDD-CPIDD-N

Cell extracts

+ ++

+

+++

+

PIDD-C

PIDD-CC

Figure 1 PIDD is constitutively processed. (A) Detection of endogenous PIDD. Western blot analysis of cell extracts of various cell lines probedwith a monoclonal anti-PIDD antibody recognizing the death domain of PIDD. (B) Schematic representation of the PIDD fragments andcleavage sites. PIDD is processed at Ser446 giving rise to the PIDD-C fragment and at Ser588 resulting in the PIDD-CC fragment. FL, full-length;DD, death domain; ZU-5, ZU-5 domain (domain present in ZO-1 and Unc5-like netrin receptors); ID: intermediate domain; LRR, leucine-richrepeat. Immunoblot analysis of extracts of HEK293Tcells stably expressing PIDD shows the various fragments. (C) Mutants at the cleavage siteare no longer processed. Mutant forms of PIDD were stably expressed in HEK293T cells and cell extracts were analyzed by Western blotting.(D) HEK293T cells stably expressing PIDD with an N-terminal VSV and a C-terminal Flag tag were lysed and anti-VSV and anti-Flagimmunoprecipitates analyzed by Western blots. (E) HEK293T cells were transiently transfected with the indicated constructs and an anti-VSVimmunoprecipitation was performed. PIDD-N is VSV-tagged and PIDD-C and PIDD-CC constructs are Flag-tagged.

Autoproteolysis of PIDD shapes its functionA Tinel et al

The EMBO Journal VOL 26 | NO 1 | 2007 &2007 European Molecular Biology Organization198

cell line, but was approximately 2:1 in most cases. As PIDD is

a p53-inducible gene, we investigated whether p53 influences

the extent of PIDD processing. Although both fragments were

generated in the presence and absence of p53, the amount

of PIDD-C appeared to be slightly higher in p53-proficient

cells (Figure 1A).

In order to map the two processing sites, C-terminally Flag-

tagged PIDD was immunoprecipitated from human embryo-

nic kidney 293T (HEK293T) cells stably expressing PIDD. The

purified protein was trypsinized and subjected to tandem

mass spectrometry. Protein fragments detected in the digests

of PIDD-C and PIDD-CC and not in PIDD-FL were identified as

SWFLVVSR and SWYWLWYTTK, respectively. This allowed

us to map the sites as F445/S446 (PIDD-C) and F587/S588

(PIDD-CC) (Figure 1B). The mapping of the cleavage sites

was confirmed by mutational analysis, as S446A and S588A

mutations inhibited the generation of the different fragments

(Figure 1C). PIDD S446A was still cleaved at the S588 site and

PIDD S588A was also cleaved at the S446 site. However,

a substantial amount of PIDD-FL was detected in cells stably

expressing the PIDD S446A mutant, but not PIDD S588A

mutant, suggesting that the processing was not as efficient

in the PIDD S446A mutant as in PIDD wildtype (wt) or PIDD

S588A mutant (see also further).

Importantly, PIDD fragments remained associated after

processing (Figure 1D). Immunoprecipitates of a PIDD con-

struct containing both an N-terminal VSV and a C-terminal

Flag tag revealed that a considerable portion of the respective

fragments remained bound to each other (Figure 1D). PIDD-N

appears to bind equally to PIDD-C and PIDD-CC (Figure 1E).

Additional mapping experiments indicated that the intra-

molecular interaction was mediated by the regions flanking

the two ZU-5 domains (Supplementary Figure 1C–E).

PIDD cleavage occurs by auto-processing

A close examination of the sequence surrounding the two

cleavage sites revealed high sequence similarity, suggesting

that one distinct protease was responsible for both PIDD

processing events (Figure 2A). Interestingly, the two surroun-

ding sequences also showed strong similarity to a sequence

found in Nup98 (Figure 2A), a component of the nuclear pore

(Fabre et al, 1994; Fontoura et al, 1999). Nup98 contains a

highly conserved HFS motif, which corresponds to the site of

auto-processing that is essential for the assembly of Nup98

in the nuclear pore complex (Rosenblum and Blobel, 1999;

Hodel et al, 2002). The proposed self-cleavage mechanism for

Nup98 is similar to that of other auto-proteolytic enzymes

including the self-splicing protein inteins, hedgehog, EMR2

and the N-terminal nucleophile (NTN) family of enzymes

(Krasnoperov et al, 1997; Paulus, 2000; Perler and Adam,

2000; Nechiporuk et al, 2001; Chang et al, 2003). All classes

of enzymes utilize acyl shift chemistry, with the side chains

of cysteine, threonine or serine acting as the nucleophile.

In addition, the inteins and the NTN enzymes contain a

conserved histidine residue positioned two residues N-terminal

from the catalytic C/T/S side chain (Figure 2A). Analysis of

the Nup98 structure revealed that the HFS motif resides in a

hydrophobic pocket, which is closed by a loop composed of

residues N-terminal to the HFS motif (Hodel et al, 2002). This

results in the requirement of a sequence of approximately

120 aa at the N-terminus of the HFS motif but only a short

sequence (approximately 8 aa) at the C-terminus of Nup98 for

successful auto-processing. Mutants of PIDD with deletions

leaving around 40–60 aa on each side from the S446

site (PIDDD1–379 and PIDDD490–554) were generated and

stably expressed in HEK293T cells (Figure 2B). Only mutant

PIDDD490–554 was processed at S446, indicating that for

auto-processing, a longer sequence N-terminal to the HFS

motif and a shorter C-terminal region are required, as is the

case with Nup98. In agreement with this result, PIDD-CC was

not generated in mutant PIDDD490–554, strengthening the fact

that both sites are processed by the same unusual mechanism.

The sequence resemblance between Nup98 and the two

PIDD cleavage sites therefore suggested that auto-processing

of PIDD was a more likely mechanism than cleavage by

exogenous proteases. Self-proteolysis reactions preceding

serines, cysteines or threonines involve a nucleophilic attack

by the hydroxyl or thiol group of the respective amino acids

on the preceding peptide bond (Rosenblum and Blobel,

1999), resulting in the replacement of the peptide bond by

an ester or a thioester bond (Figure 2C). These bonds are

more reactive than peptide bonds and can then be attacked

by a second nucleophile and broken. This model implies that

serine, cysteine or threonine (in the case of PIDD, a serine) is

essential for the reaction, and that they are interchangeable

with only limited effects on catalytic activity. In contrast,

nonhydroxyl-containing amino acids are predicted to inacti-

vate the enzymatic activity. As expected, mutating the active

site S446 or S588 to Ala completely inhibited the generation

of the PIDD-C and PIDD-CC fragments, respectively, whereas

mutating S446 and S588 to cysteine still permitted cleavage

and almost equivalent amounts of the PIDD-C or PIDD-CC

fragment were detectable (Figure 2D). The importance of the

conserved HFS motif was further investigated by mutating

F445 to Trp or His. Both mutations led to the ablation of the

activity, indicating delicate structural requirements (the ana-

logous Phe-Trp change in Nup98 conserves the activity). In

agreement with the proposed role of the His in the HFS motif,

acting to deprotonate the OH group of Ser (Figure 2C),

replacement of H444 with Gln resulted in inactivation of

the proteolytic activity (Figure 2D, left panel). Analogous

mutations in the second HFS motif also led to the disappear-

ance of the PIDD-CC fragment (Figure 2D, right panel). To

definitively prove that cleavage of the PIDD precursor is a

self-catalyzed process, we purified PIDD from HEK293T cells

that stably expressed PIDD mutants unable to spontaneously

generate the PIDD-C fragment. On the basis of mutations in

Nup98 shown to hydrolyze very slowly in the absence of

exogenously added nucleophiles (Rosenblum and Blobel,

1999), the purified non-cleavable mutants H444Q/S446C

and F445H were exposed to hydroxylamine (NH2OH),

which in both cases caused auto-processing as evidenced

by the appearance of the PIDD-C fragment (Figure 2E and

Supplementary Figure 2). Processing was not observed in the

presence of denaturing sodium dodecyl sulfate (SDS) or with

the S446A mutant, indicating that cleavage induced by

NH2OH indeed occurred at the S446 site. Taken together,

the above results indicate that PIDD is one of the few known

human proteins where auto-processing occurs in an intein-

like manner.

PIDD-N, a regulatory fragment

In order to investigate the functional consequences, if at all,

of PIDD processing, we expressed the individual fragments

Autoproteolysis of PIDD shapes its functionA Tinel et al

&2007 European Molecular Biology Organization The EMBO Journal VOL 26 | NO 1 | 2007 199

on their own and measured their capacity to interact with

molecules known to be present in the PIDDosome. We first

concentrated on the PIDD-N fragment. Unlike PIDD-FL

or PIDD-C, PIDD-N did not interact with RAIDD, and thus

did not activate caspase-2 (Figure 3A). Similarly, PIDD-N-

expressing cells did not significantly alter NF-kB activation

in response to DNA damage (Figure 3B). In contrast, cells

expressing a PIDD mutant lacking the N-terminal LRR

showed enhanced activation of NF-kB (Figure 3B). These

data suggest that PIDD-N somehow represses PIDD functions

and that its deletion leads to increased activation of PIDD.

This is reminiscent of proteins of other initiator caspase-acti-

vating platforms, such as the apoptosome (Hu et al, 1998;

Srinivasula et al, 1998), and the NOD-like receptor family,

A

E

PIDD S446PIDD S588

Nup98

435

577

454

596

853 872

R L W A H C Q V P H F S W F L V V S R PH L Y A R F Q V T H F S W Y W L W Y

DT T

T G S W V F K V S H F S K Y G L Q S D

h / 2 2

+NH2OH

SDS

/ 2 2

+ +++++++

PIDDH444Q S446C

PIDDS446A

PIDDF445H

WB: αPIDD

PIDD-C

PIDD-FL

PIDD-CC

Induction ofPIDD mutant

5 hIP: αFlag Elution Flag

peptideAdd NH2OH± SDS 2%

Incubation RT

2 2 / 2 22

B

D

C

WB: αFlag

WB: αRAIDD

WT

S44

6A

S44

6C

F44

5H

F44

5W

H44

4Q

Moc

k

∆428

–43

4

PIDD-C

PIDD-FL

PIDD-CC

WT

S58

8A

S58

8C

F58

7H

F58

2A

S44

6A/S

588A

WB: αFlag

WB: αRAIDD

Moc

k

PID

D

PID

D∆1

–379

PID

D∆4

90–5

54

83 -

47 -

32 -

Unprocessed PIDD∆1–379

Processed PIDD∆490–554

62 -

PIDD

PIDDPIDD

PIDD

PIDDPIDD

His444

His444

Ser446

Ser446

Phe445

Phe445

His444Ser446

Phe445

O

O

OO

OO

O

O

O

O

HOHO

H2N

H2N

OH2

OH

N N

N

N

N

N

NN

N

N

H

NH

NNH

H

H

H

Figure 2 PIDD undergoes auto-processing at Ser446 and Ser588. (A) Alignment of amino-acid sequences surrounding the two PIDD cleavagesites together with the Nup98 cleavage site. The arrow indicates the cleavage site. (B) Deletion mutants of PIDD (D1–379 and D490–554) or thefull-length protein were stably expressed in HEK293T cells and processing was analyzed by Western blotting. (C) Proposed mechanism for theautoproteolytic process of PIDD at the PIDD-C cleavage site, based on the mechanism proposed by Rosenblum and Blobel (1999). An identicalmechanism is also proposed for the PIDD-CC cleavage site. (D) PIDD-FL containing mutations in amino acids preceding or following the PIDD-C (left panel) or PIDD-CC (right panel) cleavage sites was analyzed for its capacity to auto-process. (E) In vitro processing of inactive H444Q/S446C, S446A and F445H mutants induced by the nucleophile NH2OH. Expression of the Flag-tagged PIDD mutants was induced bydoxocycline treatment of HEK293Trex during 5 h and purified on an anti-Flag affinity column. Immunoprecipitates were eluted using Flagpeptides. The various purified PIDD mutants were then incubated with NH2OH in the presence or absence of denaturing SDS. PIDD cleavagewas analyzed by Western blotting using the monoclonal anti-PIDD antibody.

Autoproteolysis of PIDD shapes its functionA Tinel et al

The EMBO Journal VOL 26 | NO 1 | 2007 &2007 European Molecular Biology Organization200

such as the NODs or NALPs, where the LRRs exert a strong

auto-inhibitory effect (Poyet et al, 2001; Martinon and

Tschopp, 2005). PIDD-N, however, does not seem to act as

a dominant negative interfering with PIDD’s ability to induce

caspase-2 (Supplementary Figure 3A) or NF-kB activation

(Supplementary Figure 3B).

Cleavage at the S588 site is required for caspase-2

activation

We next evaluated the functions of the PIDD-C and PIDD-CC

fragments. We first compared the caspase-2-activating capa-

city of wt PIDD with the different mutant forms of PIDD

that are unable to generate PIDD-C, PIDD-CC or both of

these fragments. Interestingly, transient overexpression of

the PIDD-CC-deficient S588A mutant failed to activate

caspase-2, whereas the PIDD-C-deficient S446A mutant was

still competent (Figure 4A). As expected from these data, the

double-PIDD-C/PIDD-CC-deficient S446A/S588A mutant also

lost its caspase-2-activating capacity. Caspase-2 activation by

PIDD is mediated by RAIDD. Whereas wt and PIDD-C-defi-

cient S446A mutant bound to RAIDD, the PIDD-CC-deficient

S588A mutant or the double mutant did not, explaining at

the molecular level the absence of caspase-2 activation

(Figure 4B). These data suggest that the generation of the

PIDD-CC fragment is required for PIDD-induced activation of

caspase-2. To further strengthen this finding, we established

populations of HeLa cells stably expressing the different

mutants and treated them with doxorubicin (Figure 4C).

Only wt PIDD and PIDD-C-deficient S446A mutant sensitized

the cells to doxorubicin-induced apoptosis. The low level of

sensitization observed with the PIDD-CC-deficient S588A

mutant is probably owing to overexpression and residual

caspase-2 activation. In order to determine which fragment

of PIDD is the active fragment involved in caspase-2 activa-

tion, HEK293T cells stably expressing both Flag-PIDD and

VSV-RAIDD were generated. These cells exhibited constitu-

tive caspase-2 activation owing to elevated expression of

PIDD (Tinel and Tschopp, 2004). When RAIDD was immuno-

precipitated, the PIDD-CC fragment, but not PIDD-C, was

detectable in the complex (Figure 4D and Supplementary

Figure 4A), confirming the importance of PIDD-CC genera-

tion for caspase-2 activation. The PIDD-CC fragment require-

ment for activation of the caspase-2 pathway was also seen

in HEK293Trex cell lines, which express PIDD-wt, PIDD-C or

PIDD-CC in an inducible manner. Induction of expression of

the various PIDD fragments showed that caspase-2 was most

efficiently activated in the clone expressing preformed PIDD-

CC, whereas in cells expressing wt and PIDD-C, caspase-2

activation was slower and followed the appearance of the

PIDD-CC fragment (Figure 4E). Immunoprecipitation studies

performed on these cells allowed us to correlate increased

caspase-2 activation with increased RAIDD and caspase-2

recruitment (Supplementary Figure 4B). Thus, PIDD-CC

expression is necessary and sufficient for the activation of

caspase-2. Importantly, stable expression of PIDD-DD (PIDD

776–910) failed to activate caspase-2 (Supplementary Figure

4C), indicating that yet another region of PIDD is required for

caspase-2 activation in addition to PIDD-DD.

Cleavage at S446 is required for NF-jB activation

PIDD constitutes not only a platform that, upon DNA da-

mage, results in caspase-2 activation, but is also an essential

protein for the DNA-damage-triggered NF-kB response

(Janssens et al, 2005). We previously showed that, in res-

ponse to DNA damage, PIDD translocates to the nucleus

(Janssens et al, 2005). To address how PIDD processing

affects nuclear translocation, we analyzed the localization

of several processing-defective PIDD mutants upon treatment

with camptothecin (CPT), a DNA-damaging drug. When DNA

damage was induced, a substantial portion of PIDD was

detectable in the nucleus (Figure 5A and B), whereas in

non-treated cells, PIDD was mostly cytoplasmic, in agree-

ment with previous results (Janssens et al, 2005). In contrast,

a PIDD-C-deficient S446A or double S446A/S588A mutant

no longer translocated to the nucleus, whereas the lack of

cleavage before S588 in the PIDD-CC-deficient S588A mutant

only slightly affected accumulation in the nucleus upon

genotoxic stress (Figure 5A and B). These results therefore

indicate that generation of the PIDD-C fragment is a pre-

requisite for PIDD nuclear translocation upon DNA damage.

Indeed, expression of the different PIDD fragments confirmed

that PIDD-C was able to translocate to the nucleus, whereas

PIDD-CC alone showed an exclusively cytoplasmic localiza-

tion (see Supplementary Figure 5).

Moc

k

PID

D

PID

D-N

PID

D-C

Moc

k

PID

D

PID

D-N

PID

D-C

WB: αRAIDD

WB: αFlag

WB: αCasp2 Procasp2

p31

p19

PIDD-CPIDD-NPIDD-CC

No treatment Etoposide

Fol

d in

duct

ion

MockPIDDPIDD∆LRRPIDD-NPIDD-C

1

2

3

4

5

6

7

8

9

10

0

PIDD-CC

IP: αFlag LysatesA

B

Figure 3 PIDD-N is a regulatory fragment of PIDD. (A) PIDD-N isnot required for RAIDD interaction. FL PIDD, PIDD-N and PIDD-Cwere stably expressed in HEK293T cells and their interaction withRAIDD and their capacity to activate caspase-2 assayed. (B)HEK293T cells stably expressing the indicated constructs weretested for their NF-kB-activating capacity induced by DNA damage(40 mM etoposide) using a luciferase-based reporter gene assay.

Autoproteolysis of PIDD shapes its functionA Tinel et al

&2007 European Molecular Biology Organization The EMBO Journal VOL 26 | NO 1 | 2007 201

To check if the altered translocation capacity also affected

PIDD’s ability to activate NF-kB, we analyzed HEK293T cells

stably expressing the various PIDD mutants using an NF-kB-

dependent luciferase reporter gene assay (Figure 5C). We

previously demonstrated that in comparison to mock-trans-

fected cells, cells stably expressing PIDD showed a moderate

but significant enhancement of the NF-kB response upon

DNA damage (Janssens et al, 2005). In keeping with our

translocation data, we observed that the enhanced NF-kB

response was only detectable for the wt PIDD and PIDD-CC-

deficient mutant (S588A), whereas the PIDD-C-deficient

mutants (S446A and S446A/S588A) were inactive. An identical

Moc

k

PID

D

PID

DS

446A

PID

DS

588A

PID

DS

446A

/S58

8A

Moc

k

Moc

k

PID

D

PID

D

PID

DS

446A

PID

DS

446A

PID

DS

588A

PID

DS

588A

PID

DS

446A

/S58

8A

PID

DS

446A

/S58

8A

PIDD-C

PIDD-CC

PIDD-FLPIDD-C

PIDD-CC

PIDD-FL

PIDD-C

PIDD-CC

Moc

k

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IDD

PID

D

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D

VSV-RAIDDFlag-PIDD

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PIDD-CC

PIDD-C

PIDD-CC

No treatment Doxorubicin

% C

ell d

eath

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20

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0

WB: αRAIDD

WB: αRAIDD

WB: αTubulin

WB: αRAIDD

WB: αPIDD

WB: αPIDD

WB: αVSV

WB: αFADD

WB: αFlag

WB: αFlag

WB: αFlag

WB: αCasp2

WB: αCasp2

Procasp2

p31

p19

MockPIDDPIDDS446APIDDS588APIDDS446A /S588A

IP: αFlag Lysates

IP: αVSV

IP: αVSV

Lysates

WB: αVSVLysates

Procasp2

p31

p19

0h 5 8 24 0 05 58 824 24

PIDD PIDD-C PIDD-CC

A B

C

D

E

Figure 4 PIDD-CC recruits RAIDD and activates caspase-2. (A) The different PIDD mutants were transiently expressed in HEK293T cells andcaspase-2 activation determined by monitoring cleavage of caspase-2. (B) Flag-tagged PIDD mutants stably expressed in HEK293T cells andimmunoprecipitated using anti-Flag antibodies were analyzed for their capacity to interact with RAIDD. (C) HeLa cells stably expressing theindicated constructs were treated with doxorubicin (5mg/ml) and the dead cells were counted by Trypan blue. Representative of threeindependent experiments is shown. (D) HEK293T cells, stably expressing wt Flag-PIDD, VSV-RAIDD or both, were lysed and anti-VSV-RAIDDimmunoprecipitates analyzed for the presence of PIDD fragments. (E) HEK293Trex cells expressing FL-PIDD, PIDD-C or PIDD-CC were inducedby doxycycline for the indicated times and caspase-2 activation was monitored.

Autoproteolysis of PIDD shapes its functionA Tinel et al

The EMBO Journal VOL 26 | NO 1 | 2007 &2007 European Molecular Biology Organization202

tendency could be observed when phosphorylation of IkB

was used as a readout for NF-kB activation (Supplementary

Figure 6).

We next determined which fragment of PIDD interacted

with the downstream adaptor RIP1 (Figure 5D). RIP1 medi-

ates recruitment of NEMO to PIDD, resulting in NEMO

ANontreated NontreatedCPT CPT

PIDD

PIDDS588A

PIDDS446A

PIDDS446A S588A

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VSV-RAIDD ++ + +

+

++++

+

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cy nu cy nu cy nu cy nu cy cy cy cy cy cynu nu nu nu nu nu

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Figure 5 PIDD-C recruits RIP1 and activates NF-kB in response to DNA damage. (A) PIDD mutants unable to generate PIDD-C do nottranslocate to the nucleus in response to genotoxic stress. PIDD- or mutant PIDD-expressing HEK293Tcells were left untreated or stimulated for2 h with 50mM CPT. Confocal images show nuclear (blue, DRAQ5) or PIDD (green, Alexa 488) staining. (B) Subcellular fractionation of HEK293Tcells stably expressing the different PIDD mutants. The cells were treated with DMSO or with 50mM CPT. (C) PIDD-FL and PIDD mutant proteinsstably expressed in HEK293Tcells were tested for their NF-kB-activating capacity induced by DNA damage (40mM etoposide) using a luciferase-based reporter gene assay. (D) HEK293T cells were cotransfected with VSV-RAIDD or VSV-RIP1 in combination with different Flag-tagged PIDDconstructs and immunoprecipitated with an anti-VSV antibody. The presence of the different PIDD fragments in the eluates was monitored.

Autoproteolysis of PIDD shapes its functionA Tinel et al

&2007 European Molecular Biology Organization The EMBO Journal VOL 26 | NO 1 | 2007 203

sumoylation and subsequent NF-kB activation (Janssens

et al, 2005). In contrast to RAIDD that interacted with

PIDD-CC, RIP1 recruited exclusively the PIDD-C fragment.

Thus, the generation of the PIDD-C fragment is crucial

for the activation of NF-kB but not of caspase-2. Moreover,

cleavage at S446 is a prerequisite for PIDD translocation to

the nucleus in response to DNA damage, showing an intimate

link between these two processes.

The two PIDD fragments are formed in a sequential

manner

The results of Figure 1C, in which we evaluated which

fragments were formed in the different PIDD cleavage mut-

ants, already suggested that the cleavage of the two fragments

occurred in a sequential manner. Thus, the generation of

PIDD-CC appeared much more efficient in molecules that

were initially cut at S446. We confirmed this sequential

order of events by monitoring the kinetics of PIDD fragment

formation in stable cell lines expressing PIDD in an inducible

manner (Figure 6A). HEK293 Flp-In TRex (HEK293Trex) cells

were induced to express PIDD by addition of doxycycline.

Four hours following the induction, only PIDD-C was detect-

able, whereas the PIDD-CC form was absent. Minor amounts

of PIDD-FL were also detected, but PIDD-FL levels did not

increase, confirming that PIDD-FL was directly processed

after expression. PIDD-CC was detectable only after 6–8 h.

The ratio of PIDD-CC to PIDD-C then steadily increased to

attain comparable levels 16 h after induction. The notion that

PIDD processing occurs constitutively in our stable cells was

further supported by an experiment in which protein synth-

esis was blocked with cycloheximide (CHX) (Figure 6B).

Together with PIDD-FL, the amount of PIDD-C fragment

steadily diminished, whereas the relative amount of PIDD-

CC slightly increased. That the disappearance of PIDD-C

fragment represented a genuine conversion to PIDD-CC and

not proteasome-dependent degradation was apparent based

on the observation that (1) it also disappeared in the presence

of the proteasome inhibitor MG-132 and (2) in the case of the

PIDD-CC-deficient S588A mutant, the PIDD-C fragment was

stable up to 16 h following CHX addition. Of note, PIDD-FL

was rapidly and completely processed into PIDD-C and PIDD-

N. No intermediary PIDD-N fragment (1–587) was detected

(Figure 1D and Supplementary Figure 6), suggesting that

PIDD-CC does not normally arise from PIDD-FL processing.

Addition of CHX did not result in further processing of

PIDD-N (Supplementary Figure 7). A sequential processing

of PIDD-FL to PIDD-N and PIDD-C and then to PIDD-CC

is therefore likely to occur.

That this mechanism is of physiological relevance was

supported by monitoring PIDD expression in response to

DNA damage. In agreement with previous reports (Lin et al,

2000), we found that DNA damage such as that induced by

etoposide or mitomycin C treatment led to a general increase

of PIDD expression in HCT116 cells (Figure 6C). Interestingly,

PIDD-C levels increased before PIDD-CC levels in response

to both treatments.

We then investigated the possibility that distinct doses

of DNA damage insults would differentially induce the two

PIDD fragments upon de novo synthesis. To this end, we

exposed the cells to a single dose of g-irradiation, which, in

contrast to continuous treatment with topoisomerase inhibi-

tors, allows the cells to recuperate from the DNA damage

insult. We exposed HEK293T cells to different amounts of

g-irradiation, ranging from 0.1 to 10 Gy (only the results for

0.1 and 10 Gy are depicted in the figure). As can be seen in

Figure 6D, PIDD-C was the first fragment to be induced and

was present from the 8 h time point onwards. We have never

been able to detect PIDD-FL, suggesting that the first cleavage

event is indeed constitutive. PIDD-CC increase occurred

much more slowly, the actual kinetics being strongly depen-

0 8 16 16+

Hours of CHX

PIDD-C

PIDD-FL

PIDD-CC

MG-132

PIDD

0 8 16 16+

PIDDS446A

0 8 16 16+

PIDDS588AA B

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PIDD-CC

Etoposide

72 0

Mitomycin C

12 24 480 12 24 48h

C

WB: αFlag WB: αFlag

0 4 6 8 10 16h

PIDD-C

PIDD-FL

PIDD-CC

WB: αRAIDD

WB: αRAIDD

WB: αRAIDD

WB: αPIDD

WB: αPARP

WB: αPIDD

WB: αRAIDD

D0

0.1 Gy

8 24 48 72

PIDD-C

PIDD-CC

h 0

10 Gy

8 24 48 72

PARPCleaved PARP

Figure 6 PIDD-C and PIDD-CC are induced with differential kinetics. (A) Induction of PIDD expression in HEK293Trex cells by doxycyclineduring the indicated times. Processing of PIDD was monitored by Western blot analysis of the cell extracts. (B) HEK293Tcells stably expressingthe indicated PIDD mutants were treated for 0, 8 and 16 h with 10mg/ml CHX in the presence or absence of the proteasome inhibitor MG-132(10mM). Processing of PIDD was monitored by Western blot. (C) HCT116 cells were treated with 10 mM etoposide or 10mM mitomycin C for theindicated times and PIDD expression levels were monitored. (D) HCT116 cells were exposed to a single dose of g-irradiation (0.1 or 10 Gy),whereafter the cells could recuperate. Endogenous PIDD expression levels and PARP cleavage were monitored.

Autoproteolysis of PIDD shapes its functionA Tinel et al

The EMBO Journal VOL 26 | NO 1 | 2007 &2007 European Molecular Biology Organization204

dent on the dose of treatment used. Whereas low doses of

g-irradiation, with only a mild effect on cell death, induced

significant levels of PIDD-CC after 48 h, high doses, which

induced strong apoptosis, evoked an immediate induction of

both PIDD-C and PIDD-CC (Figure 6D). Interestingly, at 72 h,

PIDD-C was no longer present, suggesting that all PIDD-C

was converted into PIDD-CC. These data therefore indicate

that both PIDD fragments are not induced with the same

kinetics upon DNA damage, and may reflect a regulated

activity of the second cleavage site in response to this

stimulus.

Discussion

Protein splicing is a form of post-translational processing that

is the protein equivalent of RNA splicing. There is now formal

evidence that these reactions exist in several protein matura-

tion pathways, including hedgehog protein maturation

(Porter et al, 1996), nucleoporin processing (Rosenblum

and Blobel, 1999; Hodel et al, 2002) and GPCR processing

(Lin et al, 2004). PIDD is constitutively cleaved into three

main fragments: PIDD-N (1–445), PIDD-C (446–910) and

PIDD-CC (588–910). Although our results do not provide

direct evidence for the exact reaction mechanism involved

in PIDD processing, they are consistent with auto-proteolysis,

similar to the previously described mechanisms, as they

include: (i) the cleavage site before a serine residue, (ii)

requirement for a hydroxyl or thiol group at the cleavage

position, and (iii) the sensitivity of in vitro cleavage of the

H444Q/S446C mutant to hydroxylamine. Thus, PIDD is auto-

processed at two sites, F445/S446 and F587/S588, that are

highly conserved and contain the HFS tripeptide.

An almost identical auto-processing site is found in Nup98,

for which the three-dimensional structure has been deter-

mined (Rosenblum and Blobel, 1999; Hodel et al, 2002).

Auto-processing of precursor Nup98 into Nup98 and the

8-kDa tail fragment results in only a small molecular

rearrangement and the interaction of the mature Nup98

with the 8-kDa tail fragment persists (Hodel et al, 2002).

Non-covalent interaction between the different fragments

generated appears to be a rule for proteins that undergo

auto-processing (Brannigan et al, 1995; Hodel et al, 2002;

Chang et al, 2003; Hsieh et al, 2003). Similarly, we found

that fragments generated by both cleavage events in PIDD

remained associated in a complex containing PIDD-N and

PIDD-C or PIDD-CC. The observation that RAIDD preferen-

tially immunoprecipitates PIDD-CC and not PIDD-C suggests

that PIDD-C and PIDD-CC may not be present simultaneously

in the same complex, or that dissociation of the different

fragments may occur upon activation. The fate of the 14-kDa

fragment (446–587) generated upon cleavage at S588 is

currently unknown.

Our data unambiguously demonstrate an important role

of PIDD processing with respect to the initiation of down-

stream signaling events. We previously demonstrated that

PIDD is implicated in the activation of both caspase-2 and

NF-kB upon DNA damage. Intriguingly, both functions

appear to be controlled by different PIDD fragments gener-

ated by autoproteolysis (Figure 7). PIDD-C is able to interact

with RIP1, thereby initiating the NEMO modifications that

are required for NF-kB activation (Janssens et al, 2005).

Interestingly, the PIDD-C-deficient S446A mutant does not

translocate into the nucleus in response to DNA damage,

which is a prerequisite for NEMO modification to occur

(Huang et al, 2003; Janssens et al, 2005; Wu et al, 2006).

Theoretically, processing at S446 may unmask a nuclear

localization site or a site that allows binding to a nuclear

shuttling protein, thereby explaining the need for the first

processing. Although the PIDD-C fragment and the PIDD-CC-

deficient S588A mutant have the capacity to translocate and

induce NF-kB activation in response to DNA damage, they

neither recruit RAIDD nor activate caspase-2. The latter

functions are apparently triggered only by the RAIDD-

interacting PIDD-CC fragment, which is generated at a later

stage. It is noteworthy that the mere presence of PIDD-C and

PIDD-CC fragments found to be constitutively expressed in

a number of cell lines is insufficient to trigger caspase-2

or NF-kB activation in the absence of DNA damage. This

suggests that under normal conditions, the activities asso-

ciated with PIDD-C and PIDD-CC fragments are suppressed

by an unknown inhibitor.

We have previously shown that PIDD can act as a switch

allowing to either activate an NF-kB-dependent cell survival

pathway or trigger apoptosis by activation of caspase-2

(Janssens et al, 2005). The strength with which these two

signaling pathways are engaged will certainly be modulated

by the presence of other regulators. However, we also pro-

pose that the regulation of these alternate responses to

different stimuli might result from modulation of PIDD

processing. Indeed, the relative ratio of PIDD-C and PIDD-

CC varies in cells exposed to different DNA-damaging stimuli.

Whereas the introduction of a limited amount of DNA breaks

initially only leads to the induction of the NF-kB-activating

fragment PIDD-C, more severe DNA damage concomitantly

induced PIDD-C and PIDD-CC, correlating with a strong

apoptotic response of the cells. Thus, the temporarily distinct

generation of the PIDD-C and PIDD-CC fragments might

govern the fate of the cell, leading by default to the activation

of a cell survival pathway and, only when the cell repair

process fails, to the activation of the pro-apoptotic caspase-2

pathway.

In conclusion, we have shown that auto-processing of

PIDD generates PIDD-C and PIDD-CC fragments that are

involved in the activation of NF-kB and caspase-2, respec-

tively. Induction of PIDD upon DNA damage together with

regulated auto-processing may function to selectively direct

signaling pathways.

Materials and methods

Cell culture and biological reagentsHEK293T cells, HEK293Trex cells (Invitrogen), U2OS humanosteosarcoma cells, HCT116 p53þ /þ and p53�/� cells (gift fromDr B Vogelstein, John Hopkins University, Baltimore, MD, USA) and293T cells stably expressing different constructs of PIDD weregrown in DMEMþGlutamax (Life Technologies), supplementedwith 10% fetal calf serum, 100 U/ml penicillin and 100mg/mlstreptomycin. Raji B cells, Ramos B cells and Jurkat T cells weregrown in RPMI (Life Technologies) containing the same supple-ments. The generation of cells stably expressing PIDD or RAIDDwas described elsewhere (Tinel and Tschopp, 2004). Camptothecin(CPT), mitomycin C, MG-132, etoposide (Eto) and Ratjadone Cwere purchased from Alexis. Cells were g-irradiated with a 137Cssource in a single pulse. Doxycyline, hydroxylamine, CHX, and Flagpeptides were obtained from Sigma. Hygromycin was purchasedfrom Invitrogen.

Autoproteolysis of PIDD shapes its functionA Tinel et al

&2007 European Molecular Biology Organization The EMBO Journal VOL 26 | NO 1 | 2007 205

Expression vectorspCR3-PIDD-Flag, pMSCV-PIDD-Flag, pCR3-RAIDD-VSV and pCR3-RIP1-VSV have been described previously (Martinon et al, 2000;Tinel and Tschopp, 2004). HEK293Trex cells expressing differentconstructs of PIDD were generated as described by the manufac-turer (Invitrogen). Briefly, PIDD constructs were subcloned in thepcDNA5/FRT vector (Invitrogen) and cotransfected with the Flprecombinase-encoding plasmid (pOG44, Invitrogen) into HEK293-Trex cells. Clones were then selected with hygromycin. All PIDDconstructs are C-terminally Flag-tagged. NFconluc, containing theluciferase reporter gene driven by a minimal NF-kB responsivepromoter, was a gift from Dr A Israel (Institut Pasteur, Paris,France). Pactbgal, containing the b-galactosidase gene under thecontrol of the b actin promoter, was obtained from Dr Inoue(Institute of Medical Sciences, Tokyo, Japan).

Transient transfections and NF-jB reporter gene assaysHEK293T cells stably expressing PIDD or PIDD mutants weretransiently transfected using the DNA–calcium phosphate precipita-tion method with 100 ng pNFconluc, 100 ng pactbgal and differentconcentrations of specific expression plasmids. The total amountof DNA was kept constant by adding empty vector up to 1mg DNAper well of a six-well plate. NF-kB activity was determined bymeasuring the luciferase activity present in cell extracts. Luciferasevalues were normalized for differences in transfection efficiency onthe basis of b-galactosidase activity in the same extracts, andexpressed as fold induction values relative to the unstimulatedempty vector control. Where needed, cells were stimulated 48 hafter transfection for 16 h with 40mM Eto or left untreated.

Mass spectrometryThe samples were loaded on an 8% polyacrylamide gel andseparated by sodium dodecyl sulfate–polyacrylamide gel electro-

phoresis (SDS–PAGE). The gel was then incubated in a solution of10% acetic acid and 40% ethanol for 30 min and stained for 15 minwith a solution of 10% acetic acid containing 0.25 g/l Coomassiebrilliant blue G 250. The gel was destained with a solution of10% acetic acid until the required coloration was reached. Bandscorresponding to full-length PIDD constructs or their cleavageproducts were excised and manually in-gel digested with trypsinaccording to a described protocol (Bienvenut et al, 1999). Trypticpeptides were recovered in the supernatant of the digestion.Samples were mixed 1:10 with a-cyano-4-hydroxycinnamic acidmatrix (Sigma) dissolved in a solution containing 50% acetonitrileand 0.1% TFA at a concentration of 5 mg/ml. A 1 ml aliquot of thesample–matrix mixture was deposited onto a single well of a 100-well, stainless-steel, matrix-assisted laser desorption ionization(MALDI) target plate and analyzed on a MALDI TOF/TOF AB 4700Proteomics Analyzer (Applied Biosystems) in both the MS andMS/MS modes.

PIDD in vitro processingHEK293T-Rex cells expressing PIDD mutants were induced for 5 hwith 0.01mg/ml doxycycline. The cells were then harvested, lysedand an anti-Flag immunoprecipitation was performed. Immuno-precipitated PIDD was eluted with Flag peptides at 100mg/ml in50 mM HEPES (pH 7.4) and 150 mM NaCl. The eluates were thenincubated in the presence of 200 mM NH2OH at room temperaturefor the indicated time.

Co-immunoprecipitation and Western blottingCo-immunoprecipitation experiments with transfected proteinswere performed in lysis buffer containing 1% NP-40, 20 mM Tris,pH 7.4, 250 mM NaCl, 1 mM EDTA, 5% glycerol and a proteaseinhibitor cocktail. After lysis, the extracts were incubated with anti-Flag antibody for 2 h. After incubation, the beads were washed four

DD

ZU

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LRR

LRR

S446

S588

Caspase-2activation

NF-κB activation

Kin

ase

NE

MO

DD

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RIP1 CA

RD

Casp2

CA

RD

PIDD-CC

RAIDD

Genotoxic stress

Inhibitor

(1) (2) (3)

DD

DD

DD

DD

DD

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-5

ZU

-5

ZU

-5

ZU

-5

ZU

-5

Figure 7 Model of PIDD maturation and its impact on the selection of signaling pathways. Upon synthesis, PIDD folds into a conformation inwhich one of the potential splicing sites, S446, is immediately active, leading to the generation of PIDD-N and PIDD-C fragments (1). Cleavageat the PIDD-C proteolytic site (2) presumably induces a slight conformational change at the PIDD-CC cleavage site, allowing the proteolyticactivity of this site to become active as well, although this second cleavage event appears to be regulated by yet another mechanism, coupled tothe severity of the DNA damage (3). The two cleavage events in PIDD lead to the generation of two fragments with distinct functions. Cleavageat S446 generates PIDD-C, which is able to bind RIP1 and NEMO and as such triggers an NF-kB response. Cleavage at S588 generates PIDD-CC,which binds to RAIDD and therefore recruits and activates caspase-2. These two signaling pathways seem to be constitutively inhibited andbecome activated only upon genotoxic stress.

Autoproteolysis of PIDD shapes its functionA Tinel et al

The EMBO Journal VOL 26 | NO 1 | 2007 &2007 European Molecular Biology Organization206

times with lysis buffer and analyzed by SDS–PAGE and immuno-blotting. The antibodies used for Western blotting were anti-phospho-IkB (Cell Signalling), anti-caspase-2 11B4 (Alexis), rabbitanti-RAIDD (Alexis) and anti-FADD (Transduction Labs). A mono-clonal anti-PIDD antibody (Anto-1) was raised against PIDD-DD(776–910) according to standard procedures. AL249, a rabbit anti-PIDD polyclonal antibody raised against a peptide covering aminoacids 148–174, was purchased from Alexis.

Confocal microscopyHEK293T cells stably expressing the different PIDD mutants werecultured on sterile glass coverslips in six-well plates and fixed with3% paraformaldehyde for 15 min. Cells were permeabilized with0.3% saponin for 10 min and treated with 2% normal goat serum(NGS)/0.5% BSA/0.1% saponin as a blocking reagent. Flagantibody (M2, Sigma) was used at a dilution of 1/500 in 0.1%saponin/0.1%BSA/PBS; a secondary Alexa 488 anti-mouse IgG1(Molecular Probes) was used at a dilution of 1/300 in 0.1%saponin/0.1%BSA/PBS. Cells were mounted in FluorSave (Calbio-chem) containing a 1/1000 dilution of DRAQ5 (Alexis) for nuclearcounterstain. Images from immunostaining were acquired by usinga Zeiss inverted laser scanning confocal microscope LSM 510 witha � 63 oil-immersion objective.

Biochemical fractionationCells were lysed in a cytosolic lysis buffer containing 10 mM HEPES,pH 7.9, 1.5 mM MgCl2, 300 mM sucrose, 0.5% NP-40, 10 mM KCl,supplemented with DTT and protease inhibitors. The lysates werecentrifuged and the supernatant constituted the cytosolic fraction.The pellet was then lysed in RIPA buffer and subsequentlysonicated. The supernatant constituted the nuclear-enrichedfraction.

Supplementary dataSupplementary data are available at The EMBO Journal Online(http://www.embojournal.org).

Acknowledgements

We thank S Hertig for technical support and H Everett, E Meylan,D Muruve, M Thome and P Schneider and the whole Tschopp groupfor discussions and critical reading of the manuscript. This workwas supported by grants from the Swiss National ScienceFoundation. SJ is supported by an EMBO fellowship and EL by anFRM fellowship.

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